Information gleaned from recent studies with single-stranded, positive-sense RNA viruses pathogenic to humans and animals (polio and alphaviruses) and insects (flock house virus;FHV) revealed that the mechanism of genome packaging in these viral systems is functionally coupled to replication. Recently our laboratory adopted a novel in vivo system referred to as Agrobacterium-mediated transient expression (agroinfiltration) to study encapsidation in plants. This system not only allowed efficient expression of viral genome components either autonomously or synchronously in plant cells, but also effectively uncoupled replication from packaging. Application of the agroinfiltration system to brome mosaic virus (BMV, a plant infecting RNA virus) allowed us to hypothesize that packaging in BMV is also functionally coupled to replication. In addition, co-expression of BMV and FHV in plant cells using agroinfiltration revealed that for specific RNA packaging to occur, synchronization of replication and transcription of coat protein (CP) mRNAs from homologous replication machinery is obligatory. This two-year exploratory project is designed to evaluate, at the sub-cellular level, the intimacy of replication to packaging. An agroinfiltration system competent to synchronously infect the same plant cell with BMV and FHV will be used through out these studies. Our working hypothesis is that translation of CP followed by virus assembly occurs very close to the sites of viral replication. Thus in Aim 1, we propose to temporally and sequentially localize and identify the sub-cellular compartment(s) where translation of CP and virus assembly of BMV and FHV occurs. In addition to the molecular and biochemical characterization, delineation of CP translation and virus assembly sites at the sub-cellular level will be investigated by electron microscopy using a novel Silver Enhancement-Controlled Sequential Immunogold technique (SECSI). BMV and FHV differentially replicate on the outer membranes of endoplasmic reticulum (ER) and mitochondria respectively. We found that packaging is non-specific when BMV CP or FHV CP was expressed either transiently or via heterologous replication. Thus, experiments outlined in Aim 2 are focused in addressing, for the packaging specificity occur, whether viral progeny RNA need to be tethered to the same membrane near which it's CP is being actively synthesized. This will be investigated by retargeting the FHV replicase complex to the ER, where the synthesis of FHV CP from genetically engineered BMV RNA will be synchronized. At the completion of the project we should know whether translation of CP and assembly of virions occur at or near the replication sites and whether tethering of viral progeny RNA to the same membrane near which it's CP is being actively synthesized is obligatory to confer packaging specificity. Results obtained from this research proposal would improve our understanding concerning the mechanism of replication-coupled packaging in RNA viruses pathogenic to humans, animals and plants. PUBLIC HEALTH RELEVANCE: RNA viruses cause serious diseases in humans, animals and crop plants of agronomic importance. Since spread of these viruses between respective host organisms is dependent of formation of infectious virions, our proposed research aims will employ a multidisciplinary approach in unraveling the mechanisms that regulate the assembly process.